The basis of protein-losing enteropathy (PLE) and how it develops are unknown and no animal models exist. We propose to develop a model system to better understand PLE and test a novel therapeutic based on clinical observations and our recent discoveries. Patients with Congenital Disorders of Glycosylation (CDG) develop PLE after environmental insults such as enteric and generalized infections. Other children with normal glycosylation develop PLE months to years after their Fontan surgery to correct congenital univentricular hearts. The variable onset suggests a combined genetic/environmental etiology. We noted that CDG patients and PLE-stricken post- Fontan patients lose heparan sulfate (HS) proteoglycans (HSPG) specifically from the basolateral surface of small intestine epithelial cells. This also occurs in post-Fontan patients. In CDG patients, HSPG normalizes again when PLE resolves. This makes enterocyte HSPG the only known molecular marker of PLE in a relevant cell type. We hypothesize that localized loss of HSPG, plus a general systemic pro-inflammatory condition in a setting of increased venous pressure causes PLE. Our preliminary data supports a robust synergism of these factors. These findings allow us to construct and test a mouse model for PLE. We will study enteric protein loss in normal and HS- deficient mice and in HS-deficient cell monolayers. In AIM 1, we determine the effects of prolonged cytokine exposure in mice and the mechanism of HS-cytokine synergism in cell culture. In AIM 2 we measure the effects of increased pressure on protein loss in normal and HS-deficient mice. AIM 3 studies the synergy of increased pressure and cytokines in normal and HS-deficient mice. In Aim 4 we test the ability of a novel modified heparin to prevent or reverse protein loss. The results will extend our understanding of how PLE develops and provide a model system to study other therapeutics. In the future, our work may help identify Fontan patients at risk for developing PLE and provide insights into new therapeutics that can be tested in our model of this enigmatic disease. PUBLIC HEALTH RELEVANCE: Losing plasma proteins through the intestine (protein-losing enteropathy, PLE) can be fatal. Children with PLE have many different inherited and acquired diseases. How and why they suddenly develop PLE is not understood, and no one has an animal model to test emerging therapies. We propose to make a mouse model of PLE and test a novel therapy developed in our laboratory. Our long-range goal is to identify at-risk patients and provide a therapy for them.